Analysis of Microsatellite Instability by Melting Peak
Analysis with BAl26 and BAl2S Specific Fluorescence
Hybridization Probes
WOLFGANG DIETMAIER*,ARNDT HARTMANN, FERDINAND HOFSTADTER
Introduction
Microsatellite instability (MSI) can be detected in about 15% of all colorectal cancers (CRC) as a result of defective mismatch repair. Almost all (>90%) CRC from
patients with hereditary non-polyposis colorectal cancers (HNPCC) show MSI due
to mutations in the hMSH2, hMLHl, and hMSH6 mismatch repair genes [1,2,3,4].
MSI can also be observed in other tumors of the HNPCC tumor spectrum, e.g., gastric, ovarian, and endometrial carcinomas. In order to identify HNPCC patients,
MSI analysis of the tumor DNA and immunohistochemical detection of mismatch
repair expression in the tumor tissue is performed as a first step, followed by
germline mutation analysis of the mismatch repair gene with loss of protein expression in the tumor tissue. In colorectal tumors, microsatellite analysis is performed
by amplification of five micro satellite markers [5,6], which are separated by gel or
capillary electrophoresis and visualized using autoradiography [1], silver staining
[7], or fluorescence techniques [8,9]. A tumor with at least two unstable markers
(2/5,40%) is defined as MSI-H (high frequency microsatellite instability) [5,6].
To avoid the need for a time-consuming electrophoresis step and to minimize
contamination risk, we have established a fast and simple method for detection of
MSI using the LightCycler. The procedure is based on a real-time amplification
and subsequent melting point analysis using LCRed640- and LCRed705-labeled
hybridization probes (HyProbes), which are complementary to the mononucleotide repeats of BAT26 and BAT25 markers, respectively. MSI can be displayed
by shifted melting peaks due to alterations of the length of repetitive sequences.
Materials
LightCycler instrument
Amplification Primers (GENSET, Paris, France)
Hybridization Probes (GENSET)
* Wolfgang Dietmaier (~) (e-mail: wolfgang.dietmaier@klinik.uni-regensburg.de)
Molecular Pathology Diagnostic Unit, University of Regensburg, Franz-Josef-StrauB-Allee 11,
93053 Regensburg, Germany
Equipment
Reagents
Analysis with BAl26 and BAl2S Specific Fluorescence
Hybridization Probes
WOLFGANG DIETMAIER*,ARNDT HARTMANN, FERDINAND HOFSTADTER
Introduction
Microsatellite instability (MSI) can be detected in about 15% of all colorectal cancers (CRC) as a result of defective mismatch repair. Almost all (>90%) CRC from
patients with hereditary non-polyposis colorectal cancers (HNPCC) show MSI due
to mutations in the hMSH2, hMLHl, and hMSH6 mismatch repair genes [1,2,3,4].
MSI can also be observed in other tumors of the HNPCC tumor spectrum, e.g., gastric, ovarian, and endometrial carcinomas. In order to identify HNPCC patients,
MSI analysis of the tumor DNA and immunohistochemical detection of mismatch
repair expression in the tumor tissue is performed as a first step, followed by
germline mutation analysis of the mismatch repair gene with loss of protein expression in the tumor tissue. In colorectal tumors, microsatellite analysis is performed
by amplification of five micro satellite markers [5,6], which are separated by gel or
capillary electrophoresis and visualized using autoradiography [1], silver staining
[7], or fluorescence techniques [8,9]. A tumor with at least two unstable markers
(2/5,40%) is defined as MSI-H (high frequency microsatellite instability) [5,6].
To avoid the need for a time-consuming electrophoresis step and to minimize
contamination risk, we have established a fast and simple method for detection of
MSI using the LightCycler. The procedure is based on a real-time amplification
and subsequent melting point analysis using LCRed640- and LCRed705-labeled
hybridization probes (HyProbes), which are complementary to the mononucleotide repeats of BAT26 and BAT25 markers, respectively. MSI can be displayed
by shifted melting peaks due to alterations of the length of repetitive sequences.
Materials
LightCycler instrument
Amplification Primers (GENSET, Paris, France)
Hybridization Probes (GENSET)
* Wolfgang Dietmaier (~) (e-mail: wolfgang.dietmaier@klinik.uni-regensburg.de)
Molecular Pathology Diagnostic Unit, University of Regensburg, Franz-Josef-StrauB-Allee 11,
93053 Regensburg, Germany
Equipment
Reagents